Proton-Binding Capacity of Staphylococcus Aureus Wall Teichoic Acid and Its Role in Controlling Autolysin Activity

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Proton-Binding Capacity of Staphylococcus Aureus Wall Teichoic Acid and Its Role in Controlling Autolysin Activity Proton-Binding Capacity of Staphylococcus aureus Wall Teichoic Acid and Its Role in Controlling Autolysin Activity Raja Biswas1, Raul E. Martinez2, Nadine Go¨ hring1, Martin Schlag3, Michaele Josten4, Guoqing Xia1, Florian Hegler2, Cordula Gekeler1, Anne-Kathrin Gleske1, Friedrich Go¨ tz3, Hans-Georg Sahl4, Andreas Kappler2, Andreas Peschel1* 1 Interfaculty Institute of Microbiology and Infection Medicine, Cellular and Molecular Microbiology, University of Tu¨bingen, Tu¨bingen, Germany, 2 Center for Applied Geoscience, Geomicrobiology, University of Tu¨bingen, Tu¨bingen, Germany, 3 Interfaculty Institute of Microbiology and Infection Medicine, Microbial Genetics, University of Tu¨bingen, Tu¨bingen, Germany, 4 Institute for Medical Microbiology, Immunology and Parasitology (IMMIP), Pharmaceutical Microbiology Unit, University of Bonn, Bonn, Germany Abstract Wall teichoic acid (WTA) or related polyanionic cell wall glycopolymers are produced by most Gram-positive bacterial species and have been implicated in various cellular functions. WTA and the proton gradient across bacterial membranes are known to control the activity of autolysins but the molecular details of these interactions are poorly understood. We demonstrate that WTA contributes substantially to the proton-binding capacity of Staphylococcus aureus cell walls and controls autolysis largely via the major autolysin AtlA whose activity is known to decline at acidic pH values. Compounds that increase or decrease the activity of the respiratory chain, a main source of protons in the cell wall, modulated autolysis rates in WTA-producing cells but did not affect the augmented autolytic activity observed in a WTA-deficient mutant. We propose that WTA represents a cation-exchanger like mesh in the Gram-positive cell envelopes that is required for creating a locally acidified milieu to govern the pH-dependent activity of autolysins. Citation: Biswas R, Martinez RE, Go¨hring N, Schlag M, Josten M, et al. (2012) Proton-Binding Capacity of Staphylococcus aureus Wall Teichoic Acid and Its Role in Controlling Autolysin Activity. PLoS ONE 7(7): e41415. doi:10.1371/journal.pone.0041415 Editor: Vance G. Fowler, Duke University Medical Center, United States of America Received August 8, 2011; Accepted June 26, 2012; Published July 23, 2012 Copyright: ß 2012 Biswas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the German Research Foundation grants TRR34 to A.P. and F.G. and SFB766 to A.P., G.X., and F.G.; by the Bacteria Materials Interaction graduate programme of the University of Tu¨bingen to A.P. and A.K.; and by a fellowship from the Humboldt Foundation to R.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: andreas.peschel@uni-tuebingen.de Introduction impact on the pH-sensitive activity of cell wall-associated enzymes such as autolysins. The bacterial cell envelope governs vital processes including Peptidoglycan is built from long glycan strands that are cross- maintenance of cell shape, cell division, and protection against linked by peptide side chains. Peptidoglycan must be continuously environmental challenges. In addition, it has a crucial role in the synthesized to maintain cell integrity and viability and to allow for respiratory energy metabolism because the cytoplasmic membrane cell division. Peptidoglycan hydrolytic autolysins are critical for harbors the electron transport chain components, which generate separating daughter cells after cell division [11,12]. The mainte- a proton gradient across the membrane that is used to generate nance of the peptidoglycan network requires a fine-tuned spatial ATP or energize transport processes [1,2]. and temporal control of autolysins to prevent suicidal cell death The majority of Gram-positive bacteria contain polyanionic cell [13]. Yet, surprisingly little is known about the control of wall glycopolymers (CWGs) in their cell envelopes, which are autolysins and the underlying regulatory principles are only covalently linked to either peptidoglycan (e.g. wall teichoic acid superficially understood. Inactivation of AtlA, the major autolysin [WTA] or teichuronic acid) or membrane glycolipids (e.g. in S. aureus, or of the corresponding AtlE of Staphylococcus epidermidis lipoteichoic acid [LTA] or succinylated lipoglycans) [3]. Several is not lethal but the mutants form huge cell clusters as a result of roles in the protection and maintenance of Gram-positive cell incomplete cell separation [14]. While studying the role AtlA in S. envelopes and in bacteria-host interaction have been assigned to aureus we observed that a WTA-deficient DtagO mutant (DtagO) CWGs after WTA or LTA-deficient mutants became available in [15] is much more susceptible to autolysis than the wild type when Staphylococcus aureus and Bacillus subtilis [4–6]. Moreover, the cell autolysins were artificially activated by washing the bacteria in envelope of B. subtilis cells has been shown to be protonated during buffer with low ionic strength [16]. We have recently demonstrat- respiratory metabolism [7,8] and the polyanionic CWGs have ed that WTA prevented AtlA binding to peptidoglycan in the been implicated in cation binding [9,10]. However, it has bacterial side walls but not in the septum where WTA appeared to remained unclear if the proton-binding capacity of WTA may be less abundant or to have an altered structure. Similar observations have been made with the cell wall-binding LysM PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e41415 WTA-Mediated Autolysin Regulation domain-containing autolysins LytF of B. subtilis [11] and Sle1 (or WTA affects autolysis via AtlA Aaa) and LytN of S. aureus [17]. Moreover, several studies have The finding that WTA has a strong impact on the abundance of documented that dissipation of the membrane proton gradient protons in the cell wall suggested that the activity of pH-sensitive leads to autolysin activation in Gram-positive bacteria [18,19] but cell-wall associated enzymes such as autolysins might be affected the molecular basis of this phenomenon has remained unclear. by the presence or absence of WTA. In line with this notion WTA Here, we show that WTA contributes substantially to the has been shown to affect the activity of autolysins [20,21]. Activity concentration of proton-binding sites in the cell wall and impacts of AtlA, the major S. aureus autolysin, has its optimal activity at on S. aureus autolysis largely via the major autolysin AtlA. neutral pH and drops strongly at pH values below 6.5 [22] Compounds that modulate the activity of the respiratory chain, suggesting that its activity might be controlled by the presence of a main source of protons in the cell wall, affected autolysis in the WTA. presence of WTA but not in its absence, which supports the notion The autolytic activity of a WTA-deficient mutant DtagO that WTA contributes to the control of autolysin activity by followed a much faster kinetic than that of wild type or governing the abundance of protons in the cell wall. complemented DtagO in the absence of Triton X-100 (Fig. 1) or in its presence as described earlier [20]. In order to analyze if Results WTA deficiency impacts on autolysis via AtlA or via activation of one of the other autolysins atlA and tagO deletions were combined The proton-binding capacity of WTA in the S. aureus cell in strain DtagODatlA. This double mutant exhibited strongly wall reduced autolysis reaching a similar level as in the WTA- We hypothesized that the polyanionic properties of WTA may containing atlA-deficient strain indicating that WTA governs contribute to the binding of protons thereby affecting the local pH autolysis largely via AtlA. in the cell wall. In addition to WTA the cell envelopes of Gram- positive bacteria contain other negatively charged residues in LTA, peptidoglycan, phospholipid head groups, and surface- associated proteins. To assess if WTA contributes significantly to the overall density of negative charges in the cell envelope, the proton-binding capacities of S. aureus wild type and isogenic WTA- deficient tagO mutant (DtagO) were determined by Fourier Transform Infrared (FTIR) spectroscopy and a whole-cell titration approach. Phosphate groups were abundant in wild type cell envelopes but below the FTIR detection limit in DtagO. Accordingly, the proton- binding sites in the mutant were 23% reduced compared to the wild type (Table 1), which confirms that WTA contributes substantially to the proton-binding capacity of the S. aureus cell envelope. DtagO complemented with a plasmid-encoded copy of tagO had the same proton-binding capacity as the wild type indicating that the difference between wild type and DtagO resulted from its inability to synthesize WTA. Since the abundance of protons in the cell envelope might affect the proton gradient across the cytoplasmic membrane the membrane potential of wild type, DtagO, and complemented mutant was measured by monitoring the distribution of the small lipophilic cation [3H]tetraphenylpho- sphonium bromide ([3H]TPP+). The three strains had similar membrane potentials around 2120 mV (Tab. 1) indicating that WTA-dependent proton binding in the cell wall hardly impacts on Figure 1. Increased autolysis of S. aureus DtagO depends on the the abundance of protons at the membrane surface. major autolysin AtlA. AtlA-expressing or deficient strains are indicated by continuous or dotted lines, respectively. Means and SEM of 3 independent experiments are shown. doi:10.1371/journal.pone.0041415.g001 Table 1. Proton-binding sites and phosphate groups in the cell envelope and membrane potential of S. aureus strains. Proton-binding site Detection of phosphate group Membrane Strain concentrations (mmol/mg bacteria) absorption bands by FTIR (cm21) potential (mV) Wild type 35.6 1.
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